Process for producing L-methionine γ-lyase crystals

ABSTRACT

A process for production of L-methionine gamma-lyase crystals by using polyethylene glycol, characterized by comprising the first step of warming a solution containing L-methionine gamma-lyase before or after addition of polyethylene glycol thereto and the second step of adding an inorganic salt.

This application is the national phase under 35 U.S.C. §371 of PCTInternational Application No. PCT/JP98/00993 which has an Internationalfiling date of Mar. 11, 1998 which designated the United States ofAmerica.

TECHNICAL FIELD

This invention relates to a process for production of crystals ofL-methionine γ-lyase useful as an antitumor agent, a process forpurification of L-methionine γ-lyase comprising the process forproduction of crystals and recombinant L-methionine γ-lyase crystalsproducable by the process for this producing system.

BACKGROUND ART

L-Methionine γ-lyase (EC 4.4.1.11) is an enzyme which requires pyridoxalphosphate as a coenzyme and catalyzes α,γ-elimination and γ-replacementof L-methionine or its derivatives and also α,β-elimination andβ-replacement of S-substituted L-cysteine or its derivatives. It wasreported that the enzyme was isolated and purified from Pseudomonasputida (Nakayama, T. et al., Anal. Biochem. 138, 421-424 (1984)).Recently, it was found that L-methionine γ-lyase has an antitumoractivity (WO94/11535). In the past, L-methionine γ-lyase could beobtained in very small quantity from Pseudomonas. putida. However,recent development of recombinant DNA technology provides a possibilityof its large quantity production (Inoue, H. et al., J. Biochem. 117,1120-1125 (1995)).

Needless to say, a drug used as a pharmaceutical preparation should bepure. In the past, L-methionine γ-lyase was extracted from cells ofPseudomonas putida and purified by a combination of ion-exchange columnchromatographies (Nakayama, T. et al., Anal. Biochem. 138, 421-424(1984), Lishko, V K et al., Protein expression and purification 4,529-533 (1993)). However, such enzyme did not have an enough purity tobe used as pharmaceutical preparations, and it was difficult to purifyit on a large scale.

As a method for production of protein crystals, a method usingpolyethylene glycol is well known. Concerning L-methionine γ-lyase, aprocedure of its crystallization was reported (Esaki, N. et al., Methodsin Enzymol. 143, 459-465 (1987)). The crystallization was performed bymixing L-methionine γ-lyase with a potassium phosphate buffer containingpolyethylene glycol and leaving the mixture at room temperature (socalled vapor diffusion method). In the paper, however, L-methionineγ-lyase of high purity which had previously been purified by columnchromatographies was used for the crystallization. Moreover, thequantity of the crystals obtained was very small (1.6 mg).

DISCLOSURE OF INVENTION

It is difficult to produce a large quantity of crystals from a proteinwhich has not been purified and contains impurities. Even though suchcrystallization succeeded, it was often impractical because the crystalsstill contain impurities. Therefore, a large scale crystallization atsuch step has hardly been attempted. This invention aims to provide aprocess for production of a large quantity of pure L-methionine γ-lyasecrystals from unpurified L-methionine γ-lyase, and a process forpurification of L-methionine γ-lyase which comprises the process forproduction.

From the result of intensive studies for the above purpose, the presentinventors have found out that a highly purified L-methionine γ-lyasecrystals can be produced in a short time by the method for production ofL-methionine γ-lyase crystals using polyethylene glycol, that is to say,the first step is the warming of a solution containing L-methionineγ-lyase before or after addition of polyethylene glycol thereto and thesecond step is the addition of an inorganic salt. Thus, the presentinvention has been accomplished.

In this invention, L-methionine γ-lyase means either or both of anatural L-methionine γ-lyase produced by a microorganism such asPseudomonas putida and a recombinant L-methionine γ-lyase prepared by arecombinant DNA technique. From the point of view for industrial massproduction, the recombinant L-methionine γ-lyase is preferred.Therefore, though the recombinant L-methionine γ-lyase (hereinafterreferred to as “rMETase”) is used for explanations in the embodiments ofthe present invention, the natural L-methionine γ-lyase can also beenused.

A solution containing L-methionine γ-lyase used in the process forproducing the crystals of the invention means any of solutions whichcontain unpurified or purified L-methionine γ-lyase. Examples of thesolution include a crude enzyme solution described in the following step1, a solution containing unpurified L-methionine γ-lyase and a solutionobtained after eliminating impurities by using polyethylene glycol.However, these examples do not limit the scope of this invention.

Additionally, cationic high molecular coagulants are used foreliminating nucleic acids or endotoxins as insoluble agglutinates bybinding the cationic groups to anion groups of nucleic acids orendotoxins. Examples of the coagulants include polyethyleneimine or acationic high molecular coagulant which mainly consists of chitosan(preferred is Kurimover I (Kurita Water Industries Ltd, Tokyo, Japan)).However, these examples do not limit the scope of this invention.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a picture showing prismatic crystals of rMETase.

FIG. 2 is a picture showing bi-pyramidal crystals of rMETase.

FIG. 3 is a drawing of a strategy for preparing a novel rMETaseexpression plasmid pMGLTrc03.

BEST MODE FOR CARRYING OUT THE INVENTION

1. Preparation of Solution Containing L-methionine γ-lyase (rMETase)

(1) Cultivation of rMETase Expression Strain

An expression plasmid, into which a structural gene of L-methionineγ-lyase is inserted, is prepared according to the method described inInoue, H. et al., J. Biochem. 117, 1120-1125 (1995). More concretely,the expression plasmid is prepared by inserting a DNA fragmentcontaining L-methionine γ-lyase gene into pKK223-3, pPL-Lambda or othervectors which have a suitable promoter for effective expression ofrMETase gene in a E.coli such as lac, tac, trp, trc, or λ PL andShine-Dalgarno (SD) sequence or into pKK233-2, pTrc99A or other ATGvectors which have a translation initiation codon ATG The expressionplasmid is introduced into a suitable host cell such as E.coli strainsHB101, JM103, JM105, JM109, C600, MV1184, DH1, DH5, DH5, α, BL21. Thus,a rMETase expression strain is obtained and cultured.

(2) Elimination of the Cell Debris

After the cultivation, the cells are disrupted by a high pressurehomogenizer (APV-Gaulin) at 20 to 55° C., preferably at about 42° C. Acationic high molecular coagulant is added to it. As the cationic highmolecular coagulant, polyethyleneimine or Kurimover I is given forexample. After adjusting the final concentration of the cationic highmolecular coagulant to 0.05-0.5% (w/v), preferably 0.1-0.2% (w/v), thecell debris is eliminated by the cationic high molecular coagulant at 5to 25° C. for 1 to 20 minutes to obtain a solution of crude rMETase.Instead of the elimination by the cationic high molecular coagulant, thecell debris can be eliminated by cell disruption using a high pressurehomogenizer, centrifugation, and heat treatment of the supernatant at 55to 65° C. for 1 to 10 minutes. The obtained crude enzyme solution issalted out by an ammonium sulfate and centrifuged. The obtainedprecipitates are dissolved in a buffer solution, preferably a phosphatebuffer solution, to obtain a solution containing unpurified L-methionineγ-lyase (rMETase).

(3) Elimination of Impurities

Subsequently, polyethylene glycol of which final concentration is from 5to 25% (W/V), preferably from about 8 to about 12% (W/V) is added to theunpurified rMETase-containing solution. The mixture is stirred at about2° C. to about 15° C., preferably 4° C. for about 10 minutes to about120 minutes, preferably about 60 minutes and centrifuged to eliminateimpurities. As the polyethylene glycol, it is preferred that its averagemolecular weight is not less than about 7,200 (eg, PEG 6000). Byaddition at this step of ammonium sulfate to make its finalconcentration 8 to 10%, preferably 10% as a saturation concentration,the solubility of rMETase becomes higher and the loss of rMETase can beminimized.

2. Production of rMETase Crystals

(1) The L-methionine γ-lyase-containing solution, preferably thesolution from which impurities are eliminated at the above describedstep 1, is warmed and polyethylene glycol is added. The warming can beperformed either before or after the addition of polyethylene glycol.The polyethylene glycol can be added before and/or after the warming. Inthis case, the addition of polyethylene glycol before the warming may bedone for the above described purpose of eliminating impurities.

The temperature after the warming is about 25° C. to about 40° C.,preferably about 30° C. to about 32° C. In general, crystallization ispromoted by refrigeration. In this invention, the crystallization ofL-methionine γ-lyase was unexpectedly promoted by warming at this stage.This is one of the most important points of this invention.

The addition of polyethylene glycol can be performed according to usualmethods for protein crystallization. In preferred embodiment,polyethylene glycol having the molecular weight not less than about7,200 is added to make its final concentration about 5 to about 25%(W/V), followed by mixing and dissolving. In the case that polyethyleneglycol is separately added before and after the warming, the totalconcentration of polyethylene glycol should be adjusted to the finalconcentration mentioned above. In the case that the rMETasecontaining-solution prepared in the step 1 is used, polyethylene glycolmay be added to reach the above described final concentration includingthe remained polyethylene glycol which has been added for elimination ofimpurities

(2) Further, an inorganic salt is added to give prismatic crystals ofrMETase. As the inorganic salt, alkaline metal salts such as sodiumchloride and potassium chloride are given for example. Especially,sodium chloride is preferred. The inorganic salt is used preferably atthe final concentration of about 20 mM to about 500 mM, more preferablyat about 100 mM to about 200 mM. The inorganic salt is added in 10minutes to two hours, preferably in 20 minutes.

The crystals can be formed from the solution without mixing. However, itis preferred to mix the solution during the above described steps (1)and (2). The solution for the crystallization is preferably maintainedat pH 7 to 8, more preferably at 7.2 to 7.5.

The crystals of rMETase can be separated by centrifugation.

The obtained rMETase crystals are of high purity and pyrogenic substancehas been decreased. The crystals could be stored under cool condition inlong term period.

Highly purified rMETase crystals containing extremely little contaminantproteins can be obtained by repeating the steps (1) and (2). The rMETaseobtained by this crystallization has the same or higher purity than thatobtained by combination of several column chromatographies withoutcrystallization. Further, purification after the crystallization of thisinvention (e.g., column chromatography such as ion-exchange columnchromatography and size exclusion column chromatography ) provideshighly purified rMETase crystals, in which the amount of other containedproteins is very little and the concentration of pyrogenic substance iscontrolled to a low level enough to obtain a permission ofpharmaceutical use, the marketing approval as drugs.

It is obvious that the crystallization method has industrial andeconomical advantages against chromatography method. Therefore, themethod of purification of L-methionine γ-lyase which includes theprocess for producing the crystals of this invention is very useful forindustrial use.

Recombinant L-methionine γ-lyase crystals which are produced by theproducing process of this invention are novel. This invention alsoprovides these novel crystals. The rMETase crystals produced accordingto this invention had the same enzymatic activity as that of the naturalprotein and the amino acid sequences were identical to each other.

This invention is explained in more detail by showing examples andreference examples which do not limit the scope of this invention.

REFERENCE EXAMPLE 1

Cultivation of rMETase Expression Strain

rMETase expression plasmid pYH301 (Inoue, H. et al, J. Biochem, 117,1120-1125 (1995)) was constructed by inserting a L-methionine γ-lyasestructural gene into expression vector pKK223-3 (Amersham PharmaciaBiotech). This plasmid was introduced into Escherichia coli JM109 to beused as a rMETase expression strain. After incubation in LB medium asseed culture, the cells were cultured for 24 hours at 37° C. in Terrificbroth (Funakoshi, Tokyo, Japan).

REFERENCE EXAMPLE 2

Preparation of Crude Enzyme Solution

300 ml of a culture broth obtained in Reference example 1 wascentrifuged. About 20 g of wet cells were collected and suspended in 140ml of a buffer solution for cell disruption (1.3 mM pyridoxal phosphate(PLP), 0.01% dithiothreitol (DTT), 100 mM sodium phosphate buffersolution (Na-PB), pH 7.2), then disrupted. The suspension after the celldisruption was centrifuged, and 146 ml of cell extract was obtained(specific activity of rMETase:20 U/mg). The cell extract was heated at60° C. for 5 minutes, cooled, and centrifuged again to give 136 ml ofclear lysate as a crude enzyme solution (specific activity of rMETase:30U/mg). Otherwise, 155 ml of the suspension after the cell disruption wastreated with polyethyleneimine at 10° C. for 5 minutes and centrifugedto give 136 ml of clear lysate as a crude enzyme solution (specificactivity of rMETase:20 U/mg).

REFERENCE EXAMPLE 3

Assay of rMETase Activity

The activity of rMETase was determined as follows. The rMETase solutionobtained in the above Reference examples was diluted with a dilutionbuffer (10 μM PLP, 1 mM EDTA.2Na.2H₂O, 0.1 g/1 (±)-dithiothreitol, 0.5g/1 Tween 80, 100 mM potassium phosphate buffer solution (K-PB, pH8.0))to prepare a dilution enzyme solution. A solution containingL-methionine as a substrate was preincubated at 37° C. for 5 minutes(25mM L-methionine, 10 μM PLP, 100 mM K-PB (pH 8.0)), to which 50 μl ofthe dilution enzyme solution was added, and the mixture was reacted at37° C. for 10 minutes. 100 μl of trichloroacetic acid (500 g/L) solution(hereinafter referred to as TCA solution) was added to terminate theenzyme reaction. The mixture of 0.8 ml of the obtained enzyme reactionsolution, 1.6 ml of an acetic acid buffer solution (1 M sodiumacetate/acetic acid, pH 5.0) and 0.60 ml of MBTH solution (1 g/L3-methyl-2-benzothiazolinone hydrazone HCl.H₂O) was reacted at 50° C.for 30 minutes, then cooled to room temperature. The amount of producedα ketobutyrate was determined by measuring the absorbance at 320 nm(E₃₂₀ sample). The reference absorbance was similarly determined as ablank (E₃₂₀ blank) by adding the dilution enzyme solution and TCAsolution vice versa. 1 Unit (U) of the enzyme was defined as the amountof the enzyme producing 1 μmol of α ketobutyrate for 1 minute.

Enzymeactivity(U/ml)=(1.15·3.00/15.74/0.05/0.80/10)·(ΔE+2(ΔE)²)=0.5480·(ΔE+2(ΔE)²)

Where, ΔE: E₃₂₀sample−E₃₂₀blank, 1.15: Quantity of enzyme reactionsolution (ml), 3.00 Quantity of MBTH reaction solution (ml), 15.74:molecular extinction coefficient of azine derivatives (mM), 0.05:Quantity of enzyme reaction sample solution (ml), 0.80: Quantity of MBTHreaction sample solution (ml), 10: Reaction time (minute))

REFERENCE EXAMPLE 4

Purification of rMETase By Column Chromatography

A crude enzyme solution was obtained by cell disruption and heattreatment according to Reference example 2. 270 ml of deioninzed waterwhich contained 10 μM PLP and 0.01% 2-mercaptoethanol (2-ME) was addedto 136 ml of the crude enzyme solution. The crude enzyme solution wasadjusted to be pH 7.2 and applied to 100 ml DEAE-TOYOPEARL 650C column(Tosoh Corp., Tokyo, Japan) which had been equilibrated with 10 mM Na-PB(pH 7.2). The column was washed with 10 mM Na-PB (pH 7.2) whichcontained 50 mM NaCl, 10 μM PLP and 0.01% 2-ME. The enzyme was elutedwith 10 mM Na-PB (pH 7.2) which contained 100 mM NaCl, 10 μM PLP and0.01% 2-ME. The specific activity of the rMETase was about 46 U/mg. 100ml of 10 mM Na-PB (pH 8.0) which contained 10 μM PLP and 0.01% 2-ME wasadded to about 100 ml of the obtained active fraction, and the mixturewas adjusted to be pH 8.0 and applied to 50 ml DEAE-Sepharose FF column(Amersham Pharmacia Biotech) which had been equilibrated with 50 mMNa-PB (pH 8.0). The column was washed with 50 mM Na-PB (pH 8.0) whichcontained 80 mM NaCl, 10 μM PLP and 0.01% 2-ME. The enzyme was elutedwith 50 mM Na-PB (pH 8.0) which contained 120 mM NaCl, 10 μM PLP and0.01% 2-ME. The specific activity of the rMETase was about 50 U/mg. Theobtained active fraction was concentrated by a membrane which has acutting size of 100 kDa (Millipore). 10 ml of the concentrated solutionwas applied to 400 ml Sephacryl S-200 HR column (Pharmacia Biotech)which had been equilibrated with 10mM Na-PB (pH 7.2) contained 10 mMPLP. The specific activity of the obtained effective active fraction ofthe rMETase was 52 U/mg. The specific activity and yield of rMETaseobtained after each purification step were shown in Table 1.

TABLE 1 Specific Activity Yield Purification Steps (U/mg) (%)Supernatant after cell disruption 20 100  Crude enzyme solution 30 85DEAE-TOYOPEARL 46 65 DEAE-Sepharose FF 50 50 Concentrated Solution 50 48Sephacryl S-200 HR 52 40

Example 1

Preparation of rMETase Crystals—(1)

58 g of solid ammonium sulfate (65% saturation) was added gradually to136 ml of the crude enzyme solution prepared according to Referenceexample 2. The solution was adjusted by aqueous ammonia to be pH 7.2during addition of ammonium sulfate. The enzyme was salted out from thesolution. The precipitates were collected by centrifugation and storedin a refrigerator. The precipitates were dissolved in 40 ml of adissolution buffer (500 μM PLP, 0.05% 2-ME and 100 mM Na-PB, pH7.2) and4 g of PEG 6,000 (10% (W/V)) was added gradually, then the mixture wasstirred at 4° C. for 60 minutes. After eliminating insoluble substancesby centrifugation, the solution was stirred at 4° C. for 16 hours. Theresultant solution was warmed to 30° C. and 0.8 g of PEG 6,000 (2%(w/v)) was added thereto with stirring to dissolve the PEG 6,000. 2 mlof 4 M NaCl solution was added to the solution with stirring. Themixture was further stirred at 30° C. for 60 minutes to produceprismatic crystals. After further stirring at 4° C. for 20 hours, theprismatic crystals of rMETase were obtained by centrifugation (FIG. 1).

Example 2

Preparation of rMETase Crystals—(2)

The prismatic crystals obtained at above Example 1 were dissolved in 40ml of a dissolution buffer. This solution was warmed at 30° C. and 4 gof PEG 6,000 (10% (W/V)) was added to the solution little by little withstirring to dissolve. 2 ml of 4 M NaCl solution was added to thesolution and the mixture was stirred at 4° C. for 20 hours. Appearedbi-pyramidal crystals were collected by centrifugation. These crystalswere dissolved in 35 ml of the dissolution buffer. After warming thesolution to 30° C., 3.5 g of PEG 6,000 (10% (W/V)) was added theretowith stirring to dissolve. 1.75 ml of 4 M NaCl solution was added withstirring and the mixture was further stirred at 4° C. for 20 hours. Thebi-pyramidal crystals were collected by centrifugation (FIG. 2).

These crystals which were obtained in the above Examples 1 and 2 werehighly purified to show a single band by SDS polyacrylamide gelelectrophoresis. It was confirmed that crystallization of this inventionshowed a high purification effect. Further, such obtained specificactivity, purity and yield were same or higher than those of the rMETasewhich obtained by the purification method combined with ion-exchangecolumn chromatographies mentioned in the above Reference example 4.

Example 3

Application of rMETase Crystals to Column Chromatography

The crystals obtained in example 2 were dissolved in 60 ml of thedissolution buffer. The solution was applied to 50 ml of DEAE-SepharoseFF (Amersham Pharmacia Biotech) column chromatography which had beenequilibrated with 20 mM Na-PB (pH 7.2). The column was washed with 20 mMNa-PB (pH 7.2) which contained 50 mM NaCl, 10 μM PLP and 0.01% 2-ME. Theenzyme was eluted with 20 mM Na-PB (pH 7.2) which contained 100 mM NaCl,10 μM PLP and 0.01% 2-ME. The specific activity of the rMETase was about52 U/mg. The obtained active fraction was concentrated to be 10 ml by amembrane which has a cutting size of 100 kDa (Millipore). 10 ml of theconcentrated solution was applied to 400 ml of Sephacryl S-200 HR(Pharmacia Biotech) column chromatography which had been equilibratedwith 10 mM Na-PB (pH 7.2) containing 10 μM PLP. The specific activity ofthe obtained effective active fraction of the rMETase was 52 U/mg.

The specific activity and yield of rMETase obtained after each operationin Examples 1 to 3 were shown in Table 2.

TABLE 2 Heat Polyethyleneimine Treatment Treatment Sp. act. Yield Sp.act. Yield Purification Step (U/mg) (%) (U/mg) (%) Example 1 Supernatantafter cell 20 100 20 100 disruption Crude enzyme solution 30 85 20 92Unpurified rMETase 34 78 28 85 solution rMETase prismatic 52 70 52 77crystals Example 2 rMETase bi-pyramidal 52 65 52 72 crystals Example 3DEAE-Sepharose FF 52 55 52 61 Concentrated solution 52 53 52 59Sephacryl S-200 HR 52 48 52 53 Sp. act.: Specific Activity

Example 4

Preparation of Novel Expression Plasmid

The rMETase gene from which the initiation codon was excluded (rMETase(-ATG)) was cloned by a PCR method using pYH301 containing rMETase geneas a template. 1 μl of 10 pmol sense primer (SEQ ID No.1: 5′-CCCGGTACCACGGCTCCAAC AAGCTCCCAG-3′), 1 μl of 10 pmol antisense primer (SEQ IDNo.2:5′-CTCGAGACGG GTTCAGGCAC TCGCCTT-3′), and 8 μl of dNTP (each 2.5mM) and a tablet of Ampli Wax™ (Perkin-Elmer Corp., Conn., USA) weremixed and the resulting mixture was heated at 77° C. for 7 minutes, thencooled at 20° C. for 3 minutes. 0.5 μl of 5 U/ml Taq DNA polymerase(Takara Shuzo Co., Kyoto, Japan) and about 1 μl of 1 mg/ml pYH301 wereadded and the quantity of the solution was filled up to 90 μl withdistilled water, then 10 μl of PCR amplification buffer which isattached to the Polymerase Kit was added.

PCR amplification was conducted with DNA Thermal Cycler (ModelPJ2000:Perkin-Elmer Corp.). The reaction was set up as 1 minute at 94°C., 1.5 minutes at 55° C., and 2 minutes at 72° C. per cycle andperformed 20 cycles. The resultant mixture was heated at 72° C. for 7minutes and placed at room temperature. The whole quantity of an aquaphase under a solidified wax layer was electrophoresed on agarose gel.The amplified fragments were recovered by a DNA recovering SystemSpinBindm™ (Takara Shuzo Co.)

2 μl of the collected DNA fragments, 1 μl of pMOSBlue T-vector (AmershamPharmacia Biotech) and 17 μl of distilled water were added toReady-To-Go™ T4 DNA ligation kit (Amersham Pharmacia Biotech) and themixture was incubated at 16° C. for 45 minutes, and then E.coli DH5strain was transformed. The transformant was incubated on an agar mediumcontaining ampicillin. A plasmid containing a rMETase gene and anampicillin resistant gene which were inserted in opposite direction wasselected from the transformants and named as LMGL/T-vector.

Next, rMETase(-ATG) of LMGL/T-vector was inserted in downstream of theinitiation codon of plasmid pATG3131 which contained trc promoter, SDsequence, initiation codon (ATG), 5SrrnBT₁T₂ terminator and tetracyclineresistant gene. At first, 5 μg of pATG3131 was digested with restrictionenzyme EcoRI, flushed by Mung Bean Nuclease and digested withrestriction enzyme XbaI, followed by electrophoresis on an agarose gelto recover 3.3 kbp of fragments by SpinBind. On the other hand, 10 μg ofLMGL/T-vector was digested with restriction enzyme KpnI, flushed by aDNA Blunting kit (Takara Shuzo Co.) and digested with restriction enzymeXbaI, followed by electrophoresis on an agarose gel to recover 1.2 kbpof fragments. These two fragments were added to Ready-To-Go™ T4 DNAligation kit and incubated at 16° C. for 45 minutes, and then E.coli JM109 was transformed. This transformant was incubated on an agar mediumcontaining tetracycline, from which a plasmid containing the objectivegene was selected and named as pMGLTrc03 (FIG. 3).

Example 5

Preparation of rMETase Crystals—(3)

rMETase expression strain was prepared by introducing rMETase expressionplasmid pMGLTrc03 into E.coli JM 109. The obtained strain was grown inLB as a preseed medium and further in LB containing 0.5% glucose as aseed medium, then cells were cultured in Terrific broth (Funakoshi)containing 4% glycerol at 28° C. for 24 hours by three 30-l jarfermentors. 57 kg of the obtained culture broth was centrifuged (AlfaLaval) to collect 19 kg of concentrated cells, to which was added 17.4kg of 100 mM Na-PB solution (pH 7.5) containing 24.57 g EDTA, 10.34 gPLP and 3.3 g DTT. The obtained suspension of cells was heated at 28° C.and subjected to a high pressure homogenizer (APV-Gaulin) to disrupt.The temperature after the disruption was 42° C. 2.18 L of 5% Kurimover Isolution was added to 36.6 kg of the obtained solution within about 5minutes and the mixture was stirred for 20 minutes. 38.8 kg of theresultant solution was centrifuged to give 37.4 kg of a crude enzymesolution . The obtained solution contained 160 g of rMETase whosespecific activity was 30 U/mg.

8.86 kg of ammonium sulfate was gradually added to the crude enzymesolution (40% saturation). The solution was adjusted by aqueous ammoniato be pH 7.2 for dissolving ammonium sulfate. The enzyme wasprecipitated by salting and stored in a refrigerator. The precipitateswere collected by a centrifugal separator (Sharples). The precipitateswere dissolved into 3.6L of a dissolution buffer (0.5 mM PLP, 0.05%2-ME, 20 mM Na-PB, pH7.2), to which was added a solution of 150 g ofammonium sulfate dissolved in 0.2 L of the dissolution buffer. Thissolution was adjusted by aqueous ammonium to be pH 7.2 and cooled to 4°C., to which was added 1.6 L of a dissolution buffer containing 540 g ofPEG 6,000 gradually, and the mixture was stirred at 4° C. for 60minutes. After eliminating insoluble substances by centrifugation, 5.4 Lof the dissolution buffer containing 650 g of PEG 6,000 was graduallyadded with stirring, and the resultant mixture was warmed to 32° C.,then 540 ml of 4 M NaCl solution was added with stirring for 20 minutes.Further stirring at 32° C. for 60 minutes produced prismatic crystals ofrMETase. Further stirring at 4° C. for 20 hours for growing the crystalsand centrifugation gave the prismatic crystals of rMETase.

Example 6

Preparation of rMETase Crystals—(4)

The prismatic crystals obtained above were dissolved in 6 L of thedissolution buffer, from which insoluble substances were eliminated bycentrifugation, then 2.4 L of the dissolution buffer containing 760 g ofPEG 6,000 was gradually added thereto and the resultant mixture waswarmed to 32° C. 420 ml of 4 M NaCl solution was added for 20 minuteswith stirring and the mixture was stirred at 32° C. for 60 minutes.Further stirring at 4° C. for 20 hours produced the bi-pyramidalcrystals of rMETase, which were collected by centrifugation anddissolved in 6 L of the dissolution buffer. 125 g of rMETase wascontained in 6.3 L of the obtained crystal-redissolution buffer and thespecific activity was 50 U/mg.

Example 7

Purification of rMETase With Column Chromatography AfterCrystallization—(2)

The crystal-redissolution solution obtained in Example 6 was subjectedto DEAE-Sepharose FF (Amersham Pharmacia Biotech.) column chromatographywhich had been equilibrated with 20 mM Na-PB (pH 7.2). The column waswashed with 20 mM Na-PB (pH 7.2) containing 0.1 mM PLP and 0.01% 2-ME.The enzyme was eluted with 20 mM Na-PB (pH 7.2) containing 120 mM NaCl,0.1 mM PLP and 0.01% 2-ME. Its specific activity was 52 U/mg. 15 L ofthe obtained active fraction was concentrated to 2.7 L by a membranewhich has a cutting size of 100 kDa (Millipore). The concentratedsolution contained 101 g of rMETase. 900 ml of the concentrated solutionwas subjected to 18 L of Sephacryl S-200 HR (Amersham Pharmacia Biotech)column chromatography equilibrated with 10 mM Na-PB (pH 7.2) containing0.01 min PLP. This operation was performed three times. The obtainedeffective active fraction contained 80 g of rMETase of which specificactivity was 52 U/mg.

The specific activity and yield of rMETase after each operation inExamples 5 to 7 were shown in Table 3.

TABLE 3 Kurimover I Treatment Sp. act. yield Purification Step (U/mg)(%) Example 5 Supernatant after cell 24 100  disruption Crude enzymesolution 25 99 Unpurified rMETase 40 89 solution rMETase prismaticcrystals 52 80 Example 6 rMETase bi-pyramidal 52 77 crystals Example 7DEAE-Sepharose FF 52 64 Concentrated solution 52 62 Sephacryl S-200 HR52 50 Sp. act: Specific Activity

2 1 30 DNA Artificial Sequence sense PCR primer 1 cccggtacca cggctccaacaagctcccag 30 2 27 DNA Artificial Sequence antisense PCR primer 2ctcgagacgg gttcaggcac tcgcctt 27

INDUSTRIAL APPLICABILITY

A method for producing L-methionine γ-lyase crystals of the presentinvention can purify L-methionine γ-lyase to the same or higher levelthan that obtained by column chromatography. The purification methodusing crystallization provides the pure objective compound economicallyand on a large scale as compared to chromatography. Therefore, themethod for producing the crystals of the present invention is useful asan industrial purification of L-methionine γ-lyase.

What is claimed is:
 1. process for producing L-methionine γ-lyasecrystals which comprises dissolving precipitate containing L-methionineγ-lyase in a buffered solution at a temperature in the range of about 2°C. to about 15° C. to form a solution containing L-methionine γ-lyase,adding polyethylene glycol to the solution and warming the solution to atemperature in the range of about 25° C. to about 40° C., and adding aninorganic salt to the warm solution to promote formation of L-methionineγ-lyase crystals.
 2. The process of claim 1, wherein said inorganic saltis sodium chloride or potassium chloride.
 3. The process of claim 1,wherein the final concentration of the inorganic salt is from about 20mM to about 500 mM.
 4. The process of claim 1, wherein said polyethyleneglycol has an average molecular weight of not less than about 7,200. 5.The process of claim 1, wherein.the final concentration of polyethyleneglycol is from about 5% (W/V) to about 25% (W/V).
 6. The process ofclaim 1, wherein warming is performed before addition of polyethyleneglycol to the L-methionine γ-lyase solution.
 7. The process of claim 1,wherein warming is performed after addition of polyethylene glycol tothe L-methionine γ-lyase solution.
 8. The process of claim 1, whereinaddition of polyethylene glycol to the L-methionine γ-lyase solution isperformed before and after warming.
 9. The process of claim 1, whichcomprises a step of eliminating impurities after addition ofpolyethylene glycol to the L-methionine γ-lyase solution and beforewarming thereof.
 10. The process of claim 9, wherein said elimination isperformed by adding polyethylene glycol in the presence of ammoniumsulfate.
 11. The process of claim 1, wherein the concentration ofL-methionine γ-lyase in the L-methionine γ-lyase-containing solution isfrom about 4 g/L to about 30 g/L.
 12. The process of claim 1, whereinthe L-methionine γ-lyase-containing solution is treated with a cationichigh molecular weight coagulant.
 13. The process of claim 12, whereinsaid cationic high molecular weight coagulant is polyethyleneimine. 14.The process of claim 12, wherein the main component of the cationic highmolecular weight coagulant is chitosan.
 15. The process of claim 12,wherein said cationic high molecular weight coagulant is Kurimover I.16. The process of claim 1, wherein a series of steps, of redissolutionof L-methionine γ-lyase, warming the solution before or after additionof or to polyethylene glycol, and adding an inorganic salt, are repeatedone or more times.
 17. The process of claim 1, wherein columnchromatography is performed after the process as claimed.
 18. A crystalof recombinant L-methionine γ-lyase which is produced by the process asclaimed in claim
 1. 19. The process of claim 16, wherein columnchromatography is performed after the process as claimed.
 20. A crystalof recombinant L-methionine γ-lyase which is produced by the process asclaimed in claim 16.